Name: rearing and injection of manduca sexta larvae to assess bacterial virulence
Uploaded: 2012-12-11T12:00:00
Description: Watch this Scientific Journal Video about Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence at JoVE.com

作者要感谢古德里奇布莱尔实验室的前任委员：萨曼莎乌节路，金佰利考尔斯，艾琳·赫伯特陈，格雷格·理查兹，梅根·梅纳德，YOUNGJIN园为发展本协议所作出的贡献。这项工作是由美国国家科学基金会的资助IOS-0950873和美国国立卫生研究院NRSA奖学金FAI084441Z。

1。昆虫卵的消毒和饲养<ol><li>准备由第一高压灭菌15克所提供的琼脂的饮食在900-1,000毫升H 2 O。立即高压灭菌后，拌入166克小麦胚芽饮食和融合，以及在实验室搅拌器。倒入一盘菜（菜）冷却，然后将其转移到铝箔的饮食，包裹得紧紧的，储存于4°C。 </li><li>抵达后，消毒M.天蛾鸡蛋与250毫升0.6％的漂白剂溶液的玻璃过滤器托架和真空烧瓶装置用一个90毫米的滤纸，偶尔搅拌2-3分钟?...

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直接注入M.烟草天蛾幼虫与昆虫病原细菌，如这里描述的，作为一个简单的和有效的手段来分析细菌的毒力。的方法，还具有很高的适应性，以适应不同的实验对象和/或条件。细菌可以在注射之前，以各种方式来制备。在X的箱子线虫 ，野生型细胞生长在营养丰富的Luria-Bertani（LB）培养基中对数期通常是最致命的，注射后30小时内杀死了大部分或所有的昆虫。固定相的细胞通常...

<table border="1"><tbody><tr><td> 试剂 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td> 90毫米滤纸</td><td> Whatman公司</td><td> 1001 090 </td><td></td></tr><tr><td>玻璃过滤器支架</td><td>密理博</td><td> XX1004700 </td><td></td></tr><tr><td> 烟草天蛾鸡蛋</td><td>卡罗来纳州生物供应</td><td> 143880 </td><td></td></tr><tr><td>舞毒蛾饮食+琼脂</td><td> MP Biomedicals </td><td> 0296029301 </td><td></td></tr><tr><td> 5.5盎司塑料容器和盖子</td><td>个杯公司</td><td> URC55-0090 PL4-0090 </td><td></td></tr><tr><td> 1盎司塑料容器和盖子</td><td> DART集装箱公司</td><td> 100PC 100PCL25 </td><td></td></tr><tr><td> 1X PBS </td><td></td&gt; <td></td><td> 137毫米的NaCl，2.7 mM KCl中，8毫摩尔的Na 2 HPO 4，1.46毫摩尔KH 2 PO 4，pH为7.4 </td></tr><tr><td>注射器</td><td>汉密尔顿</td><td> 80208 </td><td> 30号，0.375“长，点式2 </td></tr></tbody></table>

rearing and injection of manduca sexta larvae to assess bacterial virulence

烟草天蛾 ，俗称烟草天蛾，被认为是一种重要的农业害虫，取食茄科植物，包括烟草和番茄。的敏感性M.烟草天蛾幼虫，各种昆虫病原细菌物种1-5，以及丰富的信息提供有关昆虫的免疫系统6-8，悬而未决的基因组序列9它一个很好的模式生物用于研究宿主-微生物相互作用在发病机制。此外，M.烟草天蛾幼虫是比较大，且易于操作，并在实验室中保持相对于其他易感昆虫物种。他们的大尺寸也有利于有效的组织/血淋巴的提取分析宿主对感染的反应。 这里介绍的方法描述了直接注射的细菌进入血淋巴（血腔） 的影响烟草天蛾幼虫。这种方法可以使用各种细菌菌种，菌株，或突变体，通过简单地监测昆虫死亡的时间注射后的毒力特性进行分析和比较。这种方法的开发研究的致病性嗜和 Photorhabdus物种，通常与线虫载体为手段，以获得进入昆虫的。通常感染昆虫病原线虫幼虫通过自然的消化系统和呼吸系统的开口，并释放它们的共生细菌进入昆虫血淋巴（血），此后不久，10。这里描述的注射方法绕过需要为线虫矢量，从而脱开细菌和线虫的效果上的昆虫。此方法允许精确枚举传染性物质（细胞或蛋白质）内的接种物，这是不可能使用其他的现有的方法，用于分析entomopathogenesis，包括刻痕11和经口毒性实验12 <eM&gt;。此外，经口毒性试验解决引入消化系统的幼虫分泌毒素的毒力，而直接注射法解决了全细胞接种的毒力。 这里所描述的直接注射法的效用是，分析细菌致病通过监测昆虫死亡率。然而，该方法可以很容易地被扩展用于分枝杆菌感染的影响研究天蛾免疫系统。这种昆虫感染的反应通过体液免疫和细胞反应。的体液应答包括确认细菌相关的模式和各种抗微生物肽的后续生产7编码这些肽的基因的表达，可以监测直接感染后，通过RNA的提取和定量PCR 13。涉及瘤细胞对感染的反应，封装和血细胞吞噬作用的传染性病原体的6 </sup&gt;。要分析这些反应，注射昆虫可以解剖和用显微镜13日，14。

是图3中所示的昆虫死亡率测定的代表性示例。在这个实验中，与约50菌落形成单位（CFU）的野生型（ATCC19061）或减毒的突变株（LRP 13）， 嗜线虫致病杆菌的生长中期日志相组（n = 6昆虫每株）昆虫注射。昆虫观察到约72小时，和记录在每个时间点％注入昆虫还活着。在这种情况下，弱毒株呈明显的延迟杀虫，野生型菌株注射后30小时内杀死了所有6幼虫之前，任何?...

Watch this Scientific Journal Video about Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence at JoVE.com

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

这里所描述的方法采用直接注射昆虫病原细菌进入血淋巴<em&gt;烟草天蛾</em&gt;昆虫的幼虫。<em&gt; M.天蛾</em是市售和充分研究昆虫。因此，此方法是一种简单的方法来从一个或两个合作伙伴的角度分析宿主细菌的相互作用。

饲养和注射<em&gt;烟草天蛾</em&gt;幼虫评估细菌的毒力

Manduca sexta, commonly known as the tobacco hornworm, is considered a significant agricultural pest, feeding on solanaceous plants including tobacco and ...

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Immunology and Infection

Rearing and Injection of Manduca sexta Larvae to Assess Bacterial Virulence

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